Spectral properties of Vivid Colors™ fluorescent proteins

Expression of the Vivid Colors™ EmGFP and YFP fusion proteins can be detected by fluorescence microscopy or flow cytometry using standard FITC filter sets. For optimal visibility, it is recommended that you use a filter set optimized for detection within the excitation and emission ranges for each fluorescent protein (see table below).

Vivid Colors™ fluorescent protein fusion vectors

These Vivid Colors™ vectors are for generation of N-terminal or C-terminal fluorescent protein fusions to your gene-of-interest (GOI). Emerald Green (EmGFP) and Yellow (YFP) Fluorescent Protein versions are available. To create a Vivid Colors™ fusion with protein, you can employ either Gateway® or TOPO®-cloning technology. Gateway® Destination Vectors require the use of an entry clone, while the TOPO® vectors require only a PCR product of your GOI (TA Cloning®). Regardless of the cloning techniques you select, the Ultimate ORF™ collection is a comprehensive and convenient source of full-length cDNA clones for creating fusion proteins.

Co-expression of your protein with a Vivid Colors™ fluorescent protein
Express your protein in its native-protein configuration AND EmGFP from a single vector. The Vivid Colors™ pcDNA™ 6.2/ EmGFP-Bsd / V5-DEST vector allows you to rapidly identify transfected cells to look for biological activity conferred by your gene. You’ll need to use a Gateway® entry-clone version of your gene to clone into this destination (DEST) vector. The Ultimate ORF™ collection is a comprehensive and convenient source of full-length entry clones for this purpose. If you already have a clone of your protein, you can amplify it by PCR and clone it with TOPO®-technology to prepare the entry-clone.

Create your own vectors with other Vivid Colors™ fluorescent proteins
The pRSET/CFP (cyan) and pRSET/BFP (blue) vectors contain alternative color versions of fluorescent proteins derived from Aequorea victoria. These proteins are commonly used with the YFP and EmGFP for multi-color applications. Configured for bacterial expression and fluorescent protein purification, these vectors can also be used for subcloning to construct customized mammalian expression vectors.

To express your gene with alternative promoters, or with other selectable markers for stable-cell generation, consider the pTracer™ vectors series. These vectors express the Cycle 3 Green Fluorescent Protein (Cycle 3 GFP) which has spectral excitation and emission wavelengths identical to wild-type GFP. Although nearly as bright as the Vivid Colors™ FPs, you’ll need to use the proper filter set to observe the fully-enhanced brightness.

pTracer™/Bsd Expression Vectors
Choose from CMV or EF-1 a promoters for expressing your gene of interest. The pTracer™ EF vector will allow expression of your protein with carboxy-terminal V-5 and 6xHis epitope tags. The pTracer™ CMV vector is for expression of your protein without any epitope tags. These pTracer™ /Bsd Vectors express the blasticidin resistance gene as a fusion product with the Cycle 3 GFP.

pTracer™/Zeo Expression Vectors
Choose from SV40, CMV or EF-1 a promoters for expressing your gene of interest. The pTracer™ EF vector will allow expression of your protein with carboxy-terminal V-5 and 6xHis epitope tags. The pTracer™ SV40 and CMV vectors are for expression of your protein without any epitope tags. These pTracer™ /Bsd Vectors express the zeocin resistance gene as a fusion product with the Cycle 3 GFP.

Promoter Characterization
If you’re trying to characterize an unknown promoter, you’ll need a cloning and reporter system to monitor expression efficiencies from your PCR-generated mutants. The pGlow and pBlue TOPO® TA Expression Kits provide the convenience of TOPO®-cloning of your PCR products, and the option to quantify promoter activity with either Cycle 3 GFP (pGlow) or b - galactosidase (pBlue) expression.

GFP (cycle 3) TOPO® TA Expression Kits
These vectors are for cloning your PCR-amplified gene of interest to generate a fusion protein with Cycle 3 GFP fused on either the N- or C-termini. With TOPO®-cloning, only a 5-minute reaction is required to generate a clone. Following verification of orientation, the fusion protein is ready to express. The vector elements for these TOPO®-cloning vectors include a CMV promoter for high-level expression of your gene of interest and a neomycin resistance marker.

Highly specific antibodies to Aequorea victoria fluorescent proteins are available, including polyclonal anti-GFP (rabbit serum) and anti-GFP (rabbit IgG fraction), for western blot detection. These antibodies have been tested and are shown to react specifically and strongly to both Vivid Colors™ EmGFP and YFP. A number of different Molecular Probes® anti-GFP antibody conjugates is available, offering you a choice of multiple detection technologies you can employ.